Compositions and Methods for Fusion Protein Separation

ABSTRACT

The present invention relates to compositions and methods for fusion protein separation utilizing a peptide linker comprising a novel thrombin cleavage site.

This application is a Continuation of U.S. application Ser. No.11/407,336, filed Apr. 20, 2006, now allowed, which claims priority toU.S. Provisional Appl. No. 60/672,879, filed on Apr. 20, 2005, theentire contents of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions and methods for fusionprotein separation.

2. Related Art

Expression systems utilizing fusion proteins are a well-acceptedtechnology for the production of recombinant proteins. In such systems,the fusion partner facilitates the expression and purification of thedesired protein. The fusion partner is frequently used to provide a“tag” which can facilitate the subsequent purification of the fusionprotein. However, in order to recover the desired protein in its nativeform or in a pharmaceutically acceptable form, the fusion partner mustbe removed once the fusion protein is isolated. The most widely usedmethod to remove the fusion partner involves the use of specificcleavage enzymes such as thrombin, factor Xa or enterokinase (Wassenberget al., Protein Sci. 6:1718 (1997); Schlumpberger et al., Protein Sci.9:440 (2000); Zaitseva et al., Protein Sci. 5:1100 (1996)). Thisinvolves the insertion of a unique amino acid sequence that is specificfor cleavage by the cleavage enzyme between the desired protein and thefusion partner. The desired protein can be recovered by the cleavage ofthe fusion protein with the cleavage enzyme (e.g., thrombin).

Thrombin is a trypsin-like serine protease which will cleave peptidebonds using the serine amino acid. The specificity of thrombin has beenstudied by a number of investigators. The previously known thrombincleavage sites are as follows (Chang, Eur. J. Biochem. 151:217 (1985);GST gene fusion system handbook, Amersham Biosciences, Edition AA, p.88-89).

-   1) P4-P3-Pro-Arg/Lys↓P1′-P2′, wherein P3 and P4 are hydrophobic    amino acids and P1′ and P2′ are non-acidic amino acids. The    Arg/Lys↓P1′ bond is cleaved.

Examples

(SEQ ID NOS: 1-3) P4 P3 Pro Arg/Lys P1′ P2′ A Leu Val Pro Arg Gly Ser BMet Tyr Pro Arg Gly Asn C Ile Arg Pro Lys Leu Lys

-   2) P2-Arg/Lys↓P1′, wherein either P2 or P1′ is Gly. The Arg/Lys↓P1′    bond is cleaved.

Examples

P2 Arg/Lys P1′ A Ala Arg Gly B Gly Lys Ala

The most frequently used thrombin cleavage sequence isLeu-Val-Pro-Arg-Gly (SEQ ID NO:4) or Leu-Val-Pro-Arg-Gly-Ser (SEQ IDNO:1), which is derived from the sequence in bovine factor XIII (Takagiet al., Biochemistry 13:750 (1974)). Cleavage occurs at the arginineresidue, resulting in the protein of interest being extended at itsamino-terminal end by either a Gly or Gly-Ser. This thrombin cleavagesequence is also adopted in several commercially available expressionplasmids, including the pGEX series (Amersham Biosciences) and the pETseries (Novagen).

More recently, the Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:1) sequence wasfurther modified to include a glycine-rich linker containing thesequence Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:5) located immediatelybefore or after the thrombin cleavage site Leu-Val-Pro-Arg-Gly-Ser (SEQID NO:1) (Guan et al., Anal. Biochem. 192:262 (1991); Hakes et al.,Anal. Biochem. 202:293 (1992)).

While cleavage by thrombin in the currently known linker sequence regionis reasonably specific, it is not absolute. Although thrombin is areasonably specific enzyme, it can use a variety of different amino acidsequences as its cleavage site. If the target protein contains thrombincleavage sites, then the cleavage can occur at those sites, resulting inthe production of an internally cleaved protein, rather than the desiredfull length protein.

For example, when Halobacterium halobium L11 protein, which contains aninternal thrombin cleavage sequence, was expressed as a GST fusionprotein that contained the Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:1)thrombin cleavage site between the GST tag and L11, treatment withthrombin resulted in cleavage within the target protein L11, and notbetween L11 and the GST tag (Porse et al., J. Mol. Biol. 276:391(1998)). The Cdc14p protein of Saccharomyces cerevisiae also has aninternal thrombin cleavage sequence. When the Cdc14p protein wasexpressed as a GST fusion protein containing aSer-Gly-Gly-Gly-Gly-Gly-Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:6) thrombincleavage site, the fusion protein was cleaved at the internal sitewithin Cdc14p as well as the site within the thrombin cleavage linker(Taylor et al., J. Biol. Chem. 272:24054 (1997)).

The present invention provides a novel linker sequence for thrombincleavage which provides superior specificity to those known in the art.

SUMMARY OF THE INVENTION

The present invention provides a peptide linker comprising a novelthrombin cleavage site, which is useful for the recombinant productionof fusion proteins comprising a protein of interest and for separationof the protein of interest from the fusion protein. In one embodiment,the peptide linker comprises the sequence:

X1-X2-Ser-Pro-X3-X4-X5

-   wherein,-   X1 is two or more amino acid residues that are the same or different    from each other;-   X2 is a hydrophobic amino acid;-   X3 is arginine or lysine;-   X4 is alanine or glycine; and-   X5 is a non-acidic amino acid.

The present invention provides a fusion protein comprising a protein ofinterest, a fusion partner, and the peptide linker of the presentinvention interposed there between. The present invention also providesa method of separating a protein of interest from a fusion protein,comprising contacting said fusion protein with a sufficient amount ofthrombin such that cleavage of the peptide linker occurs. After thecleavage reaction occurs, the protein of interest is generated from thefusion protein. The protein of interest can be recovered using simpletechniques well known to those having ordinary skill in the art.

The present invention may be used to purify any prokaryotic oreukaryotic protein that can be expressed as the product of recombinantDNA technology in a host cell. These recombinant protein productsinclude cytokines, chemokines, hormones, receptors, enzymes, storageproteins, blood proteins, mutant proteins produced by proteinengineering techniques, or synthetic proteins.

Additionally, the present invention relates to polynucleotides encodingthe peptide linkers, polynucleotides encoding the fusion proteins,vectors containing the same, and host cells containing the vectors.

The present invention further provides methods of preparing apolynucleotide encoding a fusion protein, methods for producing a fusionprotein, and methods for producing a protein of interest.

The present invention also provides kits comprising the polynucleotidesand vectors of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of pGEX4T-hIL11(A) encoding afusion protein comprising a peptide linker of the invention (SEQ IDNO:8).

FIG. 2A shows the thrombin cleavage of GST-IL11(A).

FIG. 2B shows the thrombin cleavage of GST-IL11(A).

FIG. 3 shows the analysis of IL-11(A) by MALDI-TOF.

FIG. 4 shows the schematic representation of pGEX-Thymosinβ4 encoding afusion protein comprising a peptide linker of the invention (SEQ IDNO:8).

FIG. 5 shows the thrombin cleavage of GST-Thymosinβ4.

FIG. 6 shows the analysis of thymosin P4 by MALDI-TOF.

FIG. 7 shows the schematic representation of pGEX-IL6 encoding a fusionprotein comprising a peptide linker of the invention (SEQ ID NO:8).

FIG. 8 shows the thrombin cleavage of GST-IL6.

FIG. 9 shows the schematic representation of pGEX-PAR4 encoding a fusionprotein comprising a peptide linker of the invention (SEQ ID NO:8)

FIG. 10 shows the schematic representation of pGEX-IL11(LV−) encoding afusion protein comprising a peptide linker of the invention (SEQ IDNO:7).

FIG. 11 shows the thrombin cleavage of GST-IL11(LV−).

FIG. 12 shows the schematic representation of pMAL-c2-IL11 encoding afusion protein comprising a peptide linker of the invention (SEQ IDNO:7).

FIG. 13 shows the thrombin cleavage of MBP-IL11.

FIG. 14 shows the schematic representation of pET19b-IL11 encoding afusion protein comprising a peptide linker of the invention (SEQ IDNO:7).

FIG. 15 shows the thrombin cleavage of His-IL11.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a linker peptide comprising a thrombincleavage site that is cleavable with thrombin and the like. In oneembodiment, the peptide linker comprises the sequence:

X1-X2-Ser-Pro-X3-X4-X5

-   wherein,-   X1 is two or more amino acid residues that are the same or different    from each other;-   X2 is a hydrophobic amino acid;-   X3 is arginine or lysine;-   X4 is alanine or glycine; and-   X5 is a non-acidic amino acid.

In one embodiment, X1 is four or more amino acid residues that are thesame or different from each other. In another embodiment, X1 is no morethan 10 amino acid residues, e.g., no more than 8 amino acid residues,e.g., no more than 6 amino acid residues. For example, in variousembodiments X1 may be 2-6, 2-8, 2-10, 4-6, 4-8, or 4-10 amino acidresidues.

When the peptide linker is treated with thrombin, the cleavage occurs atthe bond between X3 and X4. Relative to the cleavage site, X1 occupiespositions 5 and 6 (P5-P6) when X1 is two amino acid residues orpositions 5 to 8 (P5-P8) when X1 is four amino acid residues. X2, Ser,Pro, X3, X4 and X5 each occupies position 4, 3, 2, 1, 1′, and 2′ (P4,P3, P2, P1, P1′, P2′), respectively. In one embodiment, X1 comprises Proand Arg. In one embodiment, X3 can be Arg or Lys. Thrombin cleavespeptide bonds when either Arg or Lys precedes the carboxyl group. Inanother embodiment, X4 can be Ala or Gly. The properties of Ala and Glyare very similar, both being small and non polar amino acids. In oneembodiment, X2 can be a hydrophobic amino acid selected from the groupconsisting of Gly, Ala, Pro, Val, Leu, Ile, Met, Phe, Tyr and Trp. In aparticular embodiment, X2 is Gly. In a further embodiment, X5 can be anon-acidic amino acid selected from the group consisting of Ser, Ala,Asn, Val, Leu, Ile, Lys, Phe, Tyr and Trp. In a particular embodiment,X5 is Ser. In one embodiment, X1 can be two or more of any amino acid.In another embodiment, X1 can be four or more of any amino acid. Theexact amino acid sequence of X1 does not have significant effects onthrombin cleavage.

In one embodiment, the peptide linker comprises the sequencePro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:7). In a further embodiment,the peptide linker comprises the sequenceLeu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:8). When a fusionprotein containing this cleavage site is treated with thrombin, theArgiAla bond within the cleavage site is cleaved. In one embodiment, thepeptide linker comprises a sequence other thanPro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:7). In another embodiment,the peptide linker comprises a sequence other thanLeu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:8).

The peptide linker sequences Pro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ IDNO:7) and Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:8) differfrom previously known thrombin cleavage sites because the P3 amino acidSer is not hydrophobic. According to previous studies, optimum thrombincleavage sites contain a hydrophobic amino acid at the P3 position(Chang, Eur. J. Biochem. 151:217 (1985)). The presence of a nonhydrophobic amino acid in the P3 position is known to prolong the timefor thrombin cleavage. Raftery et al. reported that the amino acidsequence Asn-Asn-Pro-Arg-Gly-His (SEQ ID NO:9) found in the murine MRP14protein could be the substrate of thrombin, but it was a poor substratecompared with Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:1) (Raftery et al.,Protein Expr. Purif. 15:228 (1999)). In the case of fibrinogen, which isa natural substrate of thrombin, Gly is located at the P3 position infibrinopeptides A (Gly-Gly-Val-Arg↓Gly-Pro) (SEQ ID NO:10), while Ser isfound at P3 in fibrinopeptides B (Phe-Ser-Ala-Arg↓Gly-His) (SEQ IDNO:11). Fibrinopeptides A is cleaved more rapidly than fibrinopeptides Bby thrombin (Binnie et al., Blood. 81:3186 (1993)).

The present invention provides a fusion protein comprising a protein ofinterest, a fusion partner, and the peptide linker of the presentinvention interposed there between.

The terms “fusion protein” and “chimeric protein,” as used herein, areinterchangeable and refer to polypeptides and proteins which comprise aprotein of interest, a fusion partner and a linker peptide with athrombin cleavage site interposed there between. In one embodiment, theprotein of interest is linked to the N-terminus of the peptide linkerand the fusion partner is linked to the C-terminus of the peptidelinker. In another embodiment, the protein of interest is linked to theC-terminus of the peptide linker and the fusion partner is linked to theN-terminus of the peptide linker. In a further embodiment, a fusionprotein may comprise more than one protein of interest and/or more thanone fusion partner, each separated by a peptide linker. In theseembodiments, the multiple proteins of interest may be the same ordifferent, the multiple fusion partners may be the same or different,and the multiple peptide linkers may be the same or different.

The terms “protein of interest,” “desired polypeptide,” “desiredprotein,” or “target protein,” as used herein, are interchangeable andrefer to any protein or peptide the production of which is desirable. Inone embodiment, the protein or peptide is biologically active. Examplesof proteins of interest include, but are not limited to, interleukin(IL)-11, thymosin β4, thymosin α1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-13, IL-15, IL-18, Protease-activated receptor 1 (PAR1),PAR3, PAR4, RANTES, stromal cell-derived factor-1α, monocyte chemotacticprotein, stem cell factor, FLT-3L, parathyroid hormone, thrombopoietin,epidermal growth factor, basic fibroblast growth factor, insulin-likegrowth factor, granulocyte-macrophage colony stimulating factor,granulocyte colony stimulating factor, macrophage colony stimulatingfactor, platelet-derived growth factor, transforming growth factor(TGF)-β1, tumor necrosis factor (TNF)-α, interferon (IFN)-α, IFN-γ,hepatocyte growth factor, vascular endothelial growth factor andimmunoglobulin heavy chain. In one embodiment, the protein of interestis selected from the group consisting of human IL11, thymosin β4, IL-6and PAR4. In another embodiment, the protein of interest is human IL11.

The term “fusion partner,” as used herein, refers to any protein orpeptide the inclusion of which in a fusion protein is desirable. In oneembodiment, the fusion partner imparts an improved characteristic to thefusion protein, e.g., ease of purification, stability, solubility, andthe like. In one embodiment, the fusion partner is an affinity peptide.Examples of affinity peptides include, but are not limited to,glutathione-S-transferase (GST), maltose binding protein (MBP),hexahistidine, T7 peptide, ubiquitin, Flag peptide, c-myc peptide,polyarginine, polycysteine, polyphenylalanine, BTag, galactose bindingdomain, cellulose binding domain (CBD), thioredoxin, staphylococcalprotein A, streptococcal protein G, calmodulin, beta-galactosidase,chloramphenicol acetyltransferase, S-peptide, streptavidin, His-tag, andStrep-tag.

The term “peptide linker,” as used herein, refers to a specific aminoacid sequence which comprises a thrombin cleavage site which isrecognized and cleaved by thrombin.

The term “thrombin” as used herein, refers to any form of thrombin whichis capable of cleaving the peptide linker of the invention. Thrombin mayinclude naturally occurring thrombin, recombinant thrombin, thrombinfragments, and thrombin analogs, as long as some level of cleavageactivity is retained by the protein. In one embodiment, thrombin may behuman thrombin or bovine thrombin, including naturally occurringthrombin, recombinant thrombin, or thrombin fragments and analogsderived from the human or bovine sequence.

The present invention provides a method of separating a protein ofinterest from a fusion protein comprising said protein of interest, afusion partner, and a peptide linker interposed there between, saidmethod comprising contacting said fusion protein with a sufficientamount of thrombin such that cleavage of the peptide linker occurs. Inone embodiment, the fusion protein is cleaved between X3 and X4 of thepeptide linker. The method may further involve separating the protein ofinterest from the other portion of the fusion protein, e.g., by affinitypurification or size separation.

In one embodiment, the thrombin is human thrombin and the fusion proteinis contacted with about 0.1 USP units to about 1.0 USP units ofthrombin, e.g., about 0.2 units to about 0.7 units of thrombin, e.g.,about 0.2 units to about 0.5 units of thrombin, e.g., about 0.2 units toabout 0.3 units of thrombin. In another embodiment, the thrombin ishuman thrombin and the fusion protein is contacted with thrombin forabout 5 minutes to about 1.5 hours, e.g., about 8 minutes to about 1.2hours, e.g., about 10 minutes to about 1.0 hours. In a furtherembodiment, the thrombin is human thrombin and the fusion protein iscontacted with about 0.25 units of thrombin for about 30 minutes.

In one embodiment, the thrombin is bovine thrombin and the fusionprotein is contacted with about 0.1 units to about 1.0 units ofthrombin, e.g., about 0.2 units to about 0.7 units of thrombin. Inanother embodiment, the thrombin is bovine thrombin and the fusionprotein is contacted with thrombin for about 5 minutes to about 1.5hours, e.g., about 8 minutes to about 1.2 hours, e.g., about 10 minutesto about 1.0 hours. In a further embodiment, the thrombin is bovinethrombin and fusion protein is contacted with about 0.5 units ofthrombin for about 1.0 hours.

The present invention provides a polynucleotide encoding the peptidelinker of the invention. The polynucleotide may be prepared by chemicalsynthesis or cloning.

The present invention provides a polynucleotide encoding the fusionprotein of the invention. In accordance with the present invention, apolynucleotide sequence coding for a protein of interest is isolated,synthesized or otherwise obtained and operably linked to apolynucleotide sequence coding for the linker peptide. The hybridpolynucleotide containing the gene for a desired protein operably linkedto a polynucleotide sequence encoding a linker peptide is referred to asa chimeric polynucleotide. In one embodiment, the chimericpolynucleotide is prepared by amplification, e.g., polymerase chainreaction, using primers incorporating the polynucleotide sequenceencoding the peptide linker, such that the amplification productcomprises the polynucleotide encoding the peptide sequence operablylinked to the polynucleotide encoding the protein of interest. In otherembodiments, the polynucleotides are ligated together using a ligase.The chimeric polynucleotide is then operably linked to a polynucleotideencoding a fusion partner to produce a polynucleotide encoding thefusion protein. In other embodiments, a polynucleotide encoding a fusionpartner is operably linked to a polynucleotide encoding the peptidelinker to form a chimeric polynucleotide, and the chimericpolynucleotide is then linked to a polynucleotide encoding a protein ofinterest to produce a polynucleotide encoding the fusion protein.

The term “operably linked,” as used herein to refer to twopolynucleotides that encode a protein or peptide, means that the twopolynucleotides are linked in such a manner that a continuous openreading frame exists that bridges both polynucleotides. As related toregulatory elements, the term “operably linked” refers to a regulatoryelement being linked to a polynucleotide encoding a protein or peptidein such a manner that the regulatory element exerts an effect on thetranscription and/or translation of the polynucleotide.

The present invention provides a vector (e.g., a plasmid, virus, orbacteriophage vector) comprising a polynucleotide encoding the peptidelinker. In one embodiment, the vector is an expression vector. Thevector preferably is autonomously replicable in a host cell andpreferably contains a selectable marker such as a drug resistance gene(e.g., ampicillin or tetracycline) or an auxotrophy complement gene. Inone embodiment, the vector further comprises a polynucleotide encoding afusion partner operably linked, (e.g., upstream or downstream in thesame reading frame) to the polynucleotide encoding the peptide linker.In another embodiment, the vector further comprises a polynucleotideencoding a protein of interest operably linked, (e.g., upstream ordownstream in the same reading frame) to the polynucleotide encoding thepeptide linker. In another embodiment, the vector comprises apolynucleotide encoding a fusion protein comprising a protein ofinterest, a fusion partner, and a peptide linker interposed therebetween. In one embodiment, the vector comprising the polynucleotideencoding the peptide linker further comprises one or more cloning sites,e.g., restriction enzyme recognition sites, upstream and/or downstreamof the polynucleotide encoding the peptide linker to facilitate thecloning of polynucleotides encoding proteins of interest or fusionpartners into the vector in frame with the peptide linker.

The vector provides the necessary regulatory sequences (e.g.,transcription and translation elements) to control expression of thefusion protein in a suitable host cell. The regulatory sequences mayinclude one or more of promoter regions, enhancer regions, transcriptiontermination sites, ribosome binding sites, initiation codons, splicesignals, introns, polyadenylation signals, Shine/Dalgarno translationsequences, and Kozak consensus sequences. Regulatory sequences arechosen with regard to the host cell in which the fusion protein is to beproduced. Suitable bacterial promoters include, but are not limited to,bacteriophage λ pL or pR, T6, T7, T7/lacO, lac, recA, gal, trp, ara,hut, and trp-lac. Suitable eukaryotic promoters include, but are notlimited to, PRBI, GAPDH, metallothionein, thymidine kinase, viral LTR,cytomegalovirus, SV40, or tissue-specific or tumor-specific promoterssuch as α-fetoprotein, amylase, cathepsin E, M1 muscarinic receptor, orγ-glutamyl transferase.

Fusion proteins which are to be secreted from a host cell into theculture medium or into the periplasm of the host cell may also contain asignal sequence. The signal sequence may be the fusion partner or may bein addition to the fusion partner. A polynucleotide encoding a signalsequence may be operably linked to the 5′ end of the polynucleotideencoding the fusion protein. Further, an additional peptide linker maybe inserted between the signal sequence and the rest of the fusionprotein such that the signal sequence may be removed from the fusionprotein by cleavage with thrombin. Suitable signal sequences are wellknown in the art and include, for example, MBP, GST, TRX, DsbA, and LamBfrom E. coli and α-factor from yeast.

Additional examples of suitable expression vectors are found in U.S.Pat. No. 5,814,503, which is incorporated herein by reference.

The present invention provides a method of preparing a polynucleotideencoding a fusion protein, comprising inserting a polynucleotideencoding a protein of interest into a cloning site of a vector such thatthe polynucleotide is upstream or downstream and in frame with apolynucleotide encoding the peptide linker. In a further embodiment, thepolynucleotide encoding a protein of interest is inserted into a cloningsite of a vector comprising a polynucleotide sequence encoding a peptidelinker operably linked to a polynucleotide encoding a fusion partnersuch that the polynucleotide encoding a protein of interest is upstreamor downstream and in frame with a polynucleotide encoding the peptidelinker.

The present invention provides a host cell comprising a vector of theinvention. The host cell may be any cell suitable for expression offusion proteins, including prokaryotic (e.g., bacterial) and eukaryotic(e.g., fungi, yeast, animal, insect, plant) cells. Suitable prokaryotichost cells include, but are not limited to, E. coli (e.g., strains DHS,HB101, JM109, or W3110), Bacillus, Streptomyces, Salmonella, Serratia,and Pseudomonas species. Suitable eukaryotic host cells include, but arenot limited to, COS, CHO, HepG-2, CV-1, LLC-MK₂, 3T3, HeLa, RPMI8226,293, BHK-21, Sf9, Saccharomyces, Pichia, Hansenula, Kluyveromyces,Aspergillus, or Trichoderma species.

Methods and materials for preparing recombinant vectors and transforminghost cells using the same, replicating the vectors in host cells andexpressing biologically active foreign polypeptides and proteins aredescribed in Old et al., Principles of Gene Manipulation, 2nd edition,(1981); Sambrook et al., Molecular Cloning, 3rd edition, Cold SpringHarbor Laboratory, 2001, and Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York 3rd edition, (2000), eachincorporated herein by reference. Vectors may be introduced into a hostcell by any means known in the art, including, but not limited to,transformation, calcium phosphate precipitation, electroporation,lipofection, microinjection, and viral infection.

The present invention provides a method for producing a fusion protein,comprising preparing a vector comprising a polynucleotide encoding afusion protein of the invention, delivering the vector into a host cell,culturing the host cell under conditions in which the fusion protein isexpressed, and separating the fusion protein. The method may furthercomprise contacting the separated fusion protein with thrombin to cleavethe fusion protein and separating the protein of interest.

The present invention further provides a method for producing a proteinof interest, comprising preparing a vector comprising a polynucleotideencoding a fusion protein of the invention, delivering the vector into ahost cell, culturing the host cell under conditions in which the fusionprotein is expressed, separating the fusion protein, contacting theseparated fusion protein with thrombin to cleave the fusion protein, andseparating the protein of interest.

The fusion protein may be separated from the host cell by any meansknown in the art. If the fusion protein is secreted from the host cell,the culture medium containing the fusion protein may be collected. Ifthe fusion protein is not secreted from the host cell, the cell may belysed to release the fusion protein. For example, bacterial cells may belysed by application of high pressure (e.g., with a high pressurehomogenizer) or by sonication.

Preferably, the fusion protein is separated by affinity purificationbased on the fusion partner. For example, a fusion protein comprisingGST may be separated on a glutathione-containing column and a fusionprotein comprising a hexahistidine tag may be separated on ametal-containing column.

While affinity purification methods are preferred for separation of thefusion protein, any known technique for separating proteins may be usedinstead of or in addition to affinity purification, including solventextraction, ultrafiltration, ammonium sulfate fractionation, HPLC, gelfiltration chromatography, ion exchange chromatography, hydrophobicinteraction chromatography, electrophoresis, and isoelectric focusing.

In one embodiment, the separated fusion protein may be contacted withthrombin and the cleaved protein separated by affinity chromatographysuch that the fusion partner is bound to the column and the protein ofinterest passes through and is collected. In another embodiment, thefusion protein may be separated by affinity chromatography and thencontacted with thrombin after being eluted from the affinity media. In afurther embodiment, the fusion protein may be contacted with thrombinwhile the fusion protein is still attached to the affinity media,thereby releasing the protein of interest. The concentration of thrombincan range from about 0.1 to about 100 USP units/ml, e.g., about 1 toabout 50 units/ml, e.g., about 10 units/ml. Flow rate can be adjusted to0 ml/min by stopping the pump and then maintained for about 5 to about60 min, e.g., about 10 to about 15 min.

Conditions for cleavage of the fusion protein by thrombin are well knownin the art, and typically are as follows: about pH of about 7 to about9, about 4° C. to about 37° C., substrate:enzyme ratio of about 5:1 toabout 125:1 (molar ratio), for about 1 to about 24 hours.

The protein purification techniques described above may also be used forseparation of the protein of interest following cleavage of the fusionprotein by thrombin. In one embodiment, the protein of interest issubjected to cation exchange chromatography (e.g., CM SEPHAROSE) and/oran anion exchange chromatography (e.g., Q SEPHAROSE). In one embodiment,the protein of interest is first subjected to cation exchangechromatography, and subsequently subjected to anion exchangechromatography.

For example, recombinant IL-11 protein isolated from affinitychromatography can first be purified using a cation exchange column,e.g., CM Sepharose Fast Flow medium. The column packed with the mediumcan be equilibrated with Buffer B containing 25 mM Tris-HCl. Samplecontaining IL-11 protein can be diluted about 4-fold with Buffer B andthen loaded onto the column. To wash the column, Buffer B can be loadedand then Buffer E containing 0.15 M Gly-NaOH, pH 9.5 can be applied.Target protein can be eluted with Buffer F containing 0.15 M Gly-NaOH,pH 9.5 and 0.15 M NaCl. All procedures of this step may be performed atabout 4° C. The pH value of Buffer B can range from about 7.5 to about8.5. In the cases of Buffer E and Buffer F, pH can range from about 9.2to about 9.7.

Recombinant IL-11 protein eluted from the cation exchange column can befurther purified using an anion exchange column, e.g., Q Sepharose FastFlow medium. The column packed with this medium can be equilibrated withBuffer G containing 1 M Gly-NaOH, pH 9.5. This column is re-equilibratedwith Buffer H containing 40 mM Gly-NaOH, pH 9.5. Sample obtained fromthe previous step can be diluted about 4-fold with water and then loadedonto the column. Flow-through containing the target protein can then becollected. Buffer H containing 40 mM Gly-NaOH, pH 9.5 is applied to thecolumn and the flow-through is also collected. The pH value during thisstep can range from about 9.2 to about 9.7 or about 9.5. All proceduresof this anion exchange chromatography may be performed at about roomtemperature. Endotoxin can be efficiently eliminated through theseprocedures.

The invention provides a kit comprising a polynucleotide encoding apeptide linker. In one embodiment, the kit comprises a vector comprisingthe polynucleotide encoding a peptide linker. In another embodiment, thevector is an expression vector. In one embodiment, the vector furthercomprises a polynucleotide encoding a fusion partner operably linked,e.g., upstream or downstream in the same reading frame, to thepolynucleotide encoding the peptide linker. In another embodiment, thevector further comprises a polynucleotide encoding a protein of interestoperably linked, e.g., upstream or downstream in the same reading frame,to the polynucleotide encoding the peptide linker. In anotherembodiment, the vector comprises a polynucleotide encoding a fusionprotein comprising a protein of interest, a fusion partner, and apeptide linker interposed there between. In one embodiment, the vectorcomprising the polynucleotide encoding the peptide linker furthercomprises one or more cloning sites, e.g., restriction enzymerecognition sites, upstream and/or downstream of the polynucleotideencoding the peptide linker to facilitate the cloning of polynucleotidesencoding proteins of interest or fusion partners into the vector inframe with the peptide linker. In an additional embodiment, the vectorcomprises the polynucleotide encoding the peptide linker, operablylinked to a polynucleotide encoding a fusion partner, and furthercomprising one or more cloning sites upstream or downstream of thepolynucleotide encoding the peptide linker. In one embodiment, the orderof the polynucleotides is fusion partner, peptide linker, cloning sites.In an alternative embodiment, the order of the polynucleotides iscloning site, peptide linker, fusion partner. In one embodiment, the kitcomprises multiple vectors in which each vector comprises apolynucleotide encoding the peptide linker and multiple cloning sites ina different reading frame relative to the peptide linker, such that apolynucleotide encoding a protein of interest can be inserted into oneof the vectors in frame with the peptide linker.

The kit may further comprise other agents related to the use of thevectors, e.g., buffers, restriction enzymes, ligases, phosphorylases,host cells, and the like. The kit may also comprise instructions for useof the vectors, e.g., for insertion of a polynucleotide encoding aprotein of interest or production of a fusion protein.

The following examples are illustrative, but not limiting, of themethods of the present invention. Other suitable modifications andadaptations of the variety of conditions and parameters normallyencountered in medical treatment and pharmaceutical science and whichare obvious to those skilled in the art are within the spirit and scopeof the invention.

Example 1 GST-IL11(A)

In the present example, GST-IL11(A) fusion protein was prepared.GST-IL11(A) fusion protein is composed of glutathione S-transferase(GST) and human IL-11 with a thrombin cleavage site inserted therebetween. The thrombin cleavage site for GST-IL11(A) isLeu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:8).

To produce GST-IL11(A), an E. coli expression plasmid, pGEX4T-hIL11(A),was constructed (FIG. 1). The DNA sequence encoding human IL-11(A) wasobtained by PCR and site-directed mutagenesis from human IL-11 cDNA. Thefollowing primer pairs were used for PCR.

(SEQ ID NO: 12) 5′: GGA TCC CCG CGA GCT TCC CCA GAC CCT BamHI (SEQ IDNO: 13) 3′: GTC GAC CCC TTA TCA CAG CCG AGT CTT CAG SalI

The following primer pairs were used for site-directed mutagenesis.

(SEQ ID NO: 14) 5′DN: CCA GCC ACC CCC GAA CCC GCC GGC GCC (SEQ ID NO:15) 3′DN: GGC GCC GGC GGG TTC GGG GGT GGC TGG

The 5′ primer for PCR was designed to encode Pro-Arg-Ala-Ser (SEQ IDNO:16) residues. The BamHI/SalI treated DNA fragment was cloned into theBamHI/SalI site of pGEX4T-1 plasmid, generating pGEX4T-hIL11(A). Theplasmid pGEX4T-1 originally has the thrombin cleavage siteLeu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:1) behind the GST tag. Therefore,pGEX4T-hIL11(A) has the new thrombin cleavage siteLeu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:8) betweenglutathione S-transferase and human IL-11 sequences.

The site-directed mutagenesis was performed to change the Asp155 ofwild-type IL-11 (NCBI Accession No.: AAA59132) to Asn.

The amino acid sequence of the hIL11(A) including the thrombin cleavagesite is given below:

(SEQ ID NO: 17)Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓-Ala-Ser-Pro-Asp-Pro-Arg-Ala-Glu-Leu-Asp-Ser-Thr-Val-Leu-Leu-Thr-Arg-Ser-Leu-Leu-Ala-Asp-Thr-Arg-Gln-Leu-Ala-Ala-Gln-Leu-Arg-Asp-Lys-Phe-Pro-Ala-Asp-Gly-Asp-His-Asn-Leu-Asp-Ser-Leu-Pro-Thr-Leu-Ala-Met-Ser-Ala-Gly-Ala-Leu-Gly-Ala-Leu-Gln-Leu-Pro-Gly-Val-Leu-Thr-Arg-Leu-Arg-Ala-Asp-Leu-Leu-Ser-Tyr-Leu-Arg-His-Val-Gln-Trp-Leu-Arg-Arg-Ala-Gly-Gly-Ser-Ser-Leu-Lys-Thr-Leu-Glu-Pro-Glu-Leu-Gly-Thr-Leu-Gln-Ala-Arg-Leu-Asp-Arg-Leu-Leu-Arg-Arg-Leu-Gln-Leu-Leu-Met-Ser-Arg-Leu-Ala-Leu-Pro-Gln-Pro-Pro-Pro-Asn-Pro-Pro-Ala-Pro-Pro-Leu-Ala-Pro-Pro-Ser-Ser-Ala-Trp-Gly-Gly-Ile-Arg-Ala-Ala-His-Ala-Ile-Leu-Gly-Gly-Leu-His-Leu-Thr-Leu-Asp-Trp-Ala-Val-Arg-Gly-Leu-Leu-Leu-Leu-Lys-Thr-Arg-Leu

The expression plasmid pGEX4T-hIL11(A) was transformed into E. coliBL21. Transformants were inoculated in LB Broth supplemented withampicillin (50 μg/ml final concentration), and incubated at 37° C. untilOD 600 reached 0.5. Then, isopropyl-β-D-thiogalactopyranoside (IPTG) wasadded to a final concentration of 0.4 mM to induce protein expressionand the cells were grown for an additional 3 h at 37° C. The expressionof GST-IL11 was confirmed by SDS-PAGE. The cells were harvested bycentrifugation, resuspended in 50 mM Tris-HCl buffer, pH 8.0 and lysedby sonication. Ammonium sulfate was added at a final concentration of5.6% while maintaining pH 8.0 with Tris powder. Addition of ammoniumsulfate was carried out at 4° C. . The supernatant containing targetprotein was applied to a Glutathione Sepharose 4 Fast Flow (AmershamBiosciences) affinity column pre-equilibrated with 25 mM Tris-HClbuffer, pH 8.0. Following several washes, the bound fusion protein waseluted with 25 mM Tris-HCl buffer containing 150 mM NaCl and 10 mMreduced glutathione, pH 8.0.

The purified fusion protein was digested with thrombin. One hundredmicrograms of protein samples were subjected to cleavage by 0.1 units,0.25 units or 0.5 units of bovine or human thrombin in 25 mM Tris-HClbuffer containing 150 mM NaCl, pH 8, at 20° C. Aliquots were removedfrom each reaction at various time points (10 min, 30 min, 1 h, 2 h, and5 h after reaction), and heat-inactivated by boiling for 5 min to stopthe reaction. The results were analyzed by SDS-PAGE, and visualized bystaining with Coomassie brilliant blue. The fusion protein GST-hIL11(A)gradually split into two major products, the GST fusion partner (26 kDa)and IL-11 (18.2 kDa) as the reaction progressed (FIGS. 2A and 2B), andinternal cleavage within IL-11 was not observed. The GST fusion partnerwas efficiently cleaved by both bovine and human thrombin in theconcentration ranges and the time points tested (FIGS. 2A and 2B).

Following cleavage with thrombin, the IL-11 was re-chromatographed usingGlutathione Sepharose 4 Fast Flow to remove the GST portion of thefusion protein. The N-terminal amino acid sequence of the IL-11 wasanalyzed using a Procise 491A HT protein sequencer (Applied Biosystems,USA). It was confirmed that the isolated IL11(A) has the sequenceAla-Ser-Pro-Asp-Pro-Arg-Ala-Glu-Leu-Asp-Ser-Thr-Val-Leu-Leu (SEQ IDNO:42) at its N-terminus.

Alternatively, the GST portion of the fusion protein was removed whilethe fusion protein was bound on the column containing GlutathioneSepharose 4 Fast Flow medium whose binding capacity is about 10 mg perml. The column was packed with the Glutathione Sepharose 4 Fast Flowmedium, and equilibrated with about 5 times column volume (CV) of BufferB containing 25 mM Tris-HCl, pH 8.0. After loading of supernatantobtained from bacterial lysate, the column was washed with about 20 CVof Buffer B, and then equilibrated with about 5 CV of Buffer Ccontaining 25 mM Tris-HCl, pH 8.0, and 0.15 M NaCl. Thrombin dissolvedin Buffer C was then applied at about 10 units per ml of the medium (orabout 1 unit per mg of the binding capacity of the medium).Subsequently, about 3 CV of Buffer C was applied to the column andIL11(A) detached from the fusion partner was collected. All procedureswere performed at room temperature, about 20-25° C.

To check whether any internal cleavage within IL-11 occurred, IL-11 wasfurther purified by cation exchange chromatography which was followed byanion chromatography, and analyzed by Matrix-Assisted Laser DesorptionIonization Mass Spectrometer (MALDI-TOF/MS) (Proteomics Solution I(Voyager-DE STR), Applied Biosystems). The expected molecular weight ofIL-11 was 18.26 kDa according to Compute pI/MW tool (available atau.expasy.org/tools/pi_tool.html), and an 18264 Da peak was observed(FIG. 3). From this result, it was confirmed that intact IL-11 proteinwas generated after thrombin cleavage of GST-IL11(A).

More specifically, recombinant IL-11 protein isolated from affinitychromatography was first purified using CM Sepharose Fast Flow medium.The column packed with the medium was equilibrated with Buffer B. Samplecontaining IL-11 protein was diluted about 4-fold with Buffer B and thenloaded onto the column. To wash the column, Buffer B was loaded and thenBuffer E containing 0.15 M Gly-NaOH, pH 9.5 was applied. Target proteinwas eluted with Buffer F containing 0.15 M Gly-NaOH, pH 9.5 and 0.15 MNaCl. All procedures of this step were performed at about 4° C.

Recombinant IL-11 protein eluted from the cation exchange column wasthen further purified using Q Sepharose Fast Flow medium. The columnpacked with this medium was equilibrated with Buffer G containing 1 MGly-NaOH, pH 9.5. This column was re-equilibrated with Buffer Hcontaining 40 mM Gly-NaOH, pH 9.5. Sample obtained from the previousstep was diluted about 4-fold with water and then loaded onto thecolumn. Flow-through containing the target protein was then collected.Buffer H containing 40 mM Gly-NaOH, pH 9.5 was applied to the column andthe flow-through was also collected. The pH value during this step wasabout 9.5. All procedures of this anion exchange chromatography wereperformed at about room temperature. Endotoxin was efficientlyeliminated through these procedures.

The GST-IL11(A) fusion protein contains the new thrombin cleavagesequence Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg-Ala-Ser (SEQ ID NO:8). Althoughthe Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg-Ala-Ser (SEQ ID NO: 8) sequenceincludes the previously known Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:1)sequence, thrombin cleavage occurs not at Arg↓Gly but at Arg↓Ala. Itproves that thrombin prefers Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg-Ala-Ser(SEQ ID NO:8) to Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:1).

Example 2 GST-Thymosin β4

The GST-Thymosin β4 expression plasmid, pGEX-Thymosin β4, wasconstructed by inserting a cDNA of thymosin β4 into pGEX4T-1-Kan (FIG.4). A cDNA encoding human thymosin 134 was cloned from total RNAprepared from K562 cells by reverse transcription (RT)-polymerase chainreaction (PCR). The oligonucleotide sequences for PCR were as follows:

(SEQ ID NO: 18) 5′: GGATCCCCTCGAGCTTCTGACAAACCCGATATG BamHI (SEQ ID NO:19) 3′: GTCGACTTACGATTCGCCTGCTTGCTTCTC SalI

The 5′ primer was designed to contain the coding sequence forGly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:20), so that theLeu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:8) sequence could begenerated when the PCR product was cloned into pGEX4T-1 expressionplasmid.

A 135 by cDNA product was generated by the PCR. The amplified productwas cloned into the pGEM-T Easy plasmid (Promega, Wis., USA), resultingin pGEM-Thymosin β4. Following sequence confirmation, the BamHI/SalIfragment of pGEM-Thymosin β4 was cloned into the corresponding sites ofthe pGEX4T-1-Kan vector which was prepared from pGEX4T-1 by replacingthe ampicillin resistance gene with the kanamycin resistance gene.

The amino acid sequence of thymosin β4 including the thrombin cleavagesite is given below.

(SEQ ID NO: 21)Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓-Ala-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys-Phe-Asp-Lys-Ser-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu-Lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-Ala-Gly-Glu-Ser

The expression plasmids were transformed into E. coli DH5α.Transformants were inoculated in LB Broth, and incubated at 37° C. untilOD 600 reached 0.5, and IPTG was added to a final concentration of 0.4mM. Incubation was continued for 3 h, and the cells were harvested bycentrifugation. The expression of GST-Thymosin β4 was confirmed bySDS-PAGE. The bacterial pellets were resuspended in 50 mM Tris-HClbuffer, pH 8.0 buffer and lysed by sonication. GST-Thymosin β4 waspurified by Glutathione Sepharose 4 Fast Flow from the soluble fraction.

The purified fusion proteins were digested with thrombin and analyzed bySDS-PAGE using 15% tricine separation gel. One hundred micrograms ofprotein samples were subjected to cleavage by 0.5 units thrombin in 25mM Tris-HCl buffer, pH 8.0 containing 150 mM NaCl, and 10 mM reducedglutathione, at 20° C. Aliquots were removed from each reaction atvarious time points (10 min, 30 min, 1 h, and 3 h after reaction), andheat-inactivated by boiling for 5 min to stop the reaction. The fusionprotein GST-Thymosin β4 (31 kDa) gradually disappeared, and the amountof thymosin β4 (5 kDa) increased as reaction time passed (FIG. 5).

Following cleavage with thrombin, the thymosin β4 was re-chromatographedusing Glutathione Sepharose 4 Fast Flow to remove the GST portion of thefusion protein. The N-terminal amino acid sequence of the thymosin β4was analyzed using a Procise 491A HT protein sequencer (AppliedBiosystems, USA). It was confirmed that the isolated thymosin 134 hasthe sequence Ala-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile (SEQ ID NO:22) atits N-terminus.

To check whether any internal cleavage within thymosin β4 occurred,thymosin β4 was further purified by cation exchange chromatography andanion chromatography, and analyzed by MALDI-TOF/MS (Proteomics SolutionI (Voyager-DE STR), Applied Biosystems). The expected molecular weightof thymosin β4 was 5.02 kDa according to Compute pI/MW tool (availableat au.expasy.org/tools/pi_tool.html), and a 4992 Da peak was observed(FIG. 6). From this result, it was confirmed that intact thymosin β4protein was generated after thrombin cleavage of GST-Thymosin β4.

Example 3 GST-IL6

The GST-IL6 expression plasmid, pGEX-IL6, was constructed by inserting acDNA of murine IL-6 into pGEX4T-1 (FIG. 7). A cDNA encoding murine IL-6was cloned from total RNA prepared from mouse dendritic cells by RT-PCR.The oligonucleotide sequences for PCR were as follows:

(SEQ ID NO: 23) 5′: GGA TCC CCT CGA GCT TCT TTC CCT ACT TCA BamHI (SEQID NO: 24) 3′: GTC GAC CTA GGT TTG CCG AGT AGA TCT SalI

The 5′ primer was designed to contain the coding sequence forGly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:20), so that theLeu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:8) sequence could begenerated when the PCR product was cloned into pGEX4T-1 expressionplasmid.

A 588 by cDNA product was generated by the PCR. The amplified productwas cloned into the pGEM-T Easy plasmid, resulting in pGEM-IL6.Following sequence confirmation, the BamHI/SalI fragment of pGEM-IL6 wascloned into the corresponding sites of the pGEX4T-1 vector.

The amino acid sequence of IL-6 including the thrombin cleavage site isgiven below.

(SEQ ID NO: 25)Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓-Ala-Ser-Phe-Pro-Thr-Ser-Gln-Val-Arg-Arg-Gly-Asp-Phe-Thr-Glu-Asp-Thr-Thr-Pro-Asn-Arg-Pro-Val-Tyr-Thr-Thr-Ser-Gln-Val-Gly-Gly-Leu-Ile-Thr-His-Val-Leu-Trp-Glu-Ile-Val-Glu-Met-Arg-Lys-Glu-Leu-Cys-Asn-Gly-Asn-Ser-Asp-Cys-Met-Asn-Asn-Asp-Asp-Ala-Leu-Ala-Glu-Asn-Asn-Leu-Lys-Leu-Pro-Glu-Ile-Gln-Arg-Asn-Asp-Gly-Cys-Tyr-Gln-Thr-Gly-Tyr-Asn-Gln-Glu-Ile-Cys-Leu-Leu-Lys-Ile-Ser-Ser-Gly-Leu-Leu-Glu-Tyr-His-Ser-Tyr-Leu-Glu-Tyr-Met-Lys-Asn-Asn-Leu-Lys-Asp-Asn-Lys-Lys-Asp-Lys-Ala-Arg-Val-Leu-Gln-Arg-Asp-Thr-Glu-Thr-Leu-Ile-His-Ile-Phe-Asn-Gln-Glu-Val-Lys-Asp-Leu-His-Lys-Ile-Val-Leu-Pro-Thr-Pro-Ile-Ser-Asn-Ala-Leu-Leu-Thr-Asp-Lys-Leu-Glu-Ser-Gln-Lys-Glu-Trp-Leu-Arg-Thr-Lys-Thr-Ile-Gln-Phe-Ile-Leu-Lys-Ser-Leu-Glu-Glu-Phe-Leu-Lys-Val-Thr-Leu-Arg-Ser-Thr-Arg-Gln-Thr

The expression plasmid was transformed into E. coli BL21. Transformantswere inoculated in LB Broth supplemented with ampicillin (50 μg/ml finalconcentration), and incubated at 37° C. until OD 600 reached 0.5, andIPTG was added to a final concentration of 0.4 mM. Incubation wascontinued for 3 h, and the cells were harvested by centrifugation. Theexpression of GST-IL6 was confirmed by SDS-PAGE. The bacterial pelletswere resuspended in 50 mM Tris-HCl buffer, pH 8.0 and lysed bysonication. GST-IL6 was purified by Glutathione Sepharose 4 Fast Flowfrom the soluble fraction.

The purified fusion proteins were digested with thrombin and analyzed bySDS-PAGE. One hundred micrograms of protein samples were subjected tocleavage by 0.5 units thrombin in 25 mM Tris-HCl buffer, pH 8.0containing 150 mM NaCl, and 10 mM reduced glutathione, at 20° C.Aliquots were removed from each reaction at various time points (10 min,30 min, 1 h, 2 h, and 3 h after reaction), and heat-inactivated byboiling for 5 min to stop the reaction. The fusion protein GST-IL6 (46kDa) gradually disappeared, and the amount of IL-6 (22 kDa) increased asreaction time passed (FIG. 8).

To check where the cleavage occurred by thrombin treatment, the proteinband containing IL-6 protein was obtained, and subjected to amino acidsequencing. First, the purified GST-IL6 protein was treated withthrombin for 3 h, and separated by SDS-PAGE. Then, the proteins in thepolyacrylamide gel were transferred to PVDF membrane. After the bandcontaining IL-6 protein was identified, it was sliced away from the PVDFmembrane, and subjected to amino acid sequencing. It was confirmed thatthe IL-6 has the sequence Ala-Ser-Phe-Pro-Thr-Ser-Gln-Val-Arg-Arg (SEQID NO:26) at its N-terminus.

Example 4 GST-PAR4

The protease-activated receptors (PARs) are known to be activated by theproteolysis of an N-terminal exodomain (Kahn et al., J. Clin. Invest.103:879 (1999)). There are four PARs (PAR1-4) that make up this familyof proteins, and PAR1, 3 and 4 are activated by thrombin (Coughlin,Proc. Natl. Acad. Sci. USA 96:11023 (1999)). The amino acid sequence ofthe thrombin cleavage site on PAR4 is Leu⁴³-Pro-Ala-Pro-Argi-Gly-Tyr(SEQ ID NO:27). We produced the GST-PAR4 protein which containsLeu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓-Ala-Ser (SEQ ID NO:8) sequence asanother thrombin cleavage site to test which site is preferred bythrombin.

The GST-PAR4 expression plasmid, pGEX-PAR4, was constructed by insertinga cDNA of human PAR4 into pGEX4T-1-Kan (FIG. 9). A cDNA encoding humanPAR4 was cloned from total RNA prepared from K562 cells by RT-PCR. Theoligonucleotide sequences for PCR were as follows:

(SEQ ID NO: 28) 5′: GGATCC CCT CGA GCT TCT ATG TGG GGG CGA BamHI (SEQ IDNO: 29) 3′: GTCGAC TCA GTG CAC CAG GGC CAG GTA SalI

The 5′ primer was designed to contain the coding sequence forGly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:20), so that theLeu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:8) sequence could begenerated when the PCR product was cloned into pGEX4T-1-Kan plasmid.

A 540 by cDNA product was generated by the PCR. The amplified productwas cloned into the pGEM-T Easy plasmid, resulting in pGEM-PAR4.Following sequence confirmation, the BamHI/SalI fragment of pGEM-PAR4was cloned into the corresponding sites of the pGEX4T-1-Kan vector.

The amino acid sequence of PAR4 including the thrombin cleavage site isgiven below.

(SEQ ID NO: 30)Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg↓-Ala-Ser-Met-Trp-Gly-Arg-Leu-Leu-Leu-Trp-Pro-Leu-Val-Leu-Gly-Phe-Ser-Leu-Ser-Gly-Gly-Thr-Gln-Thr-Pro-Ser-Val-Tyr-Asp-Glu-Ser-Gly-Ser-Thr-Gly-Gly-Gly-Asp-Asp-Ser-Thr-Pro-Ser-Ile-Leu-Pro-Ala-Pro-Arg-Gly-Tyr-Pro-Gly-Gln-Val-Cys-Ala-Asn-Asp-Ser-Asp-Thr-Leu-Glu-Leu-Pro-Asp-Ser-Ser-Arg-Ala-Leu-Leu-Leu-Gly-Tyr-Val-Pro-Thr-Arg-Leu-Val-Pro-Ala-Leu-Tyr-Gly-Leu-Val-Leu-Val-Val-Gly-Leu-Pro-Ala-Asn-Gly-Leu-Ala-Leu-Trp-Val-Leu-Ala-Thr-Gln-Ala-Pro-Arg-Leu-Pro-Ser-Thr-Met-Leu-Leu-Met-Asn-Leu-Ala-Thr-Ala-Asp-Leu-Leu-Leu-Ala-Leu-Ala-Leu-Pro-Pro-Arg-Ile-Ala-Tyr-His-Leu-Arg-Gly-Gln-Arg-Tyr-Pro-Phe-Gly-Glu-Ala-Ala-Cys-Arg-Leu-Ala-Thr-Ala-Ala-Leu-Tyr-Gly-His-Met-Tyr-Gly-Ser-Val-Leu-Leu-Leu-Ala-Ala-Val-Ser-Leu-Asp-Arg-Tyr-Leu-Ala-Leu-Val-HisThe internal thrombin cleavage site is underlined.

The expression plasmid is transformed into E. coli BL21. Transformantsare inoculated in LB Broth supplemented with kanamycin (50 μg/ml finalconcentration), and incubated at 37° C. until OD 600 reaches 0.5, andIPTG is added to a final concentration of 0.4 mM. Incubation iscontinued for 3 h, and the cells are harvested by centrifugation. Theexpression of GST-PAR4 is confirmed by SDS-PAGE. The bacterial pelletsare resuspended in 50 mM Tris-HCl buffer, pH 8.0 and lysed bysonication. GST-PAR4 is purified by Glutathione Sepharose 4 Fast Flowfrom the soluble fraction.

The purified fusion proteins are digested with thrombin and analyzed bySDS-PAGE. One hundred micrograms of protein samples are subjected tocleavage by 0.5 units thrombin in 25 mM Tris-HCl buffer, pH 8.0containing 150 mM NaCl, and 10 mM reduced glutathione, at 20° C.Aliquots are removed from each reaction at various time points (10 min,30 min, 1 h, 2 h, and 3 h after reaction), and heat-inactivated byboiling for 5 min to stop the reaction.

Example 5 GST-IL11(LV−)

To test whether the whole Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg-Ala-Ser (SEQID NO:8) sequence is required for efficient thrombin cleavage, anexpression plasmid encoding Pro-Arg-Gly-Ser-Pro-Arg-Ala-Ser (SEQ IDNO:7) as a thrombin cleavage site was constructed. First, the BamHI/SalIfragment containing IL-11(A) was obtained from pGEX4T-IL11(A) which wasdescribed in EXAMPLE 1. Then, it was inserted into the BamHI/SalI siteof pGEX6P-2, resulting in pGEX6P-IL11. The plasmid pGEX6P-2 contains“Pro-Leu” sequence upstream of the BamHI site. Therefore, pGEX6P-IL11can have Pro-Leu-Gly-Ser-Pro-Arg-Ala-Ser (SEQ ID NO:31) because theBamHI/SalI fragment containing IL-11(A) starts asGly-Ser-Pro-Arg-Ala-Ser (SEQ ID NO:20) sequence. The second amino acidLeu was changed to Arg by site-directed mutagenesis to producePro-Arg-Gly-Ser-Pro-Arg-Ala-Ser (SEQ ID NO:7) sequence. The followingoligonucleotides were used for the site-directed mutagenesis:

(SEQ ID NO: 32) 5′: CAG GGG CCC CGG GGA TCC CCT CGA GCT (SEQ ID NO: 33)3′: AGC TCG AGG GGA TCC CCG GGG CTG

The resulting plasmid was named as pGEX-IL11(LV−) (FIG. 10), and usedfor the production of GST-IL11(LV−).

The amino acid sequence of IL-11(LV−) including the thrombin cleavagesite is given below.

(SEQ ID NO: 34)Pro-Arg-Gly-Ser-Pro-Arg↓-Ala-Ser-Pro-Asp-Pro-Arg-Ala-Glu-Leu-Asp-Ser-Thr-Val-Leu-Leu-Thr-Arg-Ser-Leu-Leu-Ala-Asp-Thr-Arg-Gln-Leu-Ala-Ala-Gln-Leu-Arg-Asp-Lys-Phe-Pro-Ala-Asp-Gly-Asp-His-Asn-Leu-Asp-Ser-Leu-Pro-Thr-Leu-Ala-Met-Ser-Ala-Gly-Ala-Leu-Gly-Ala-Leu-Gln-Leu-Pro-Gly-Val-Leu-Thr-Arg-Leu-Arg-Ala-Asp-Leu-Leu-Ser-Tyr-Leu-Arg-His-Val-Gln-Trp-Leu-Arg-Arg-Ala-Gly-Gly-Ser-Ser-Leu-Lys-Thr-Leu-Glu-Pro-Glu-Leu-Gly-Thr-Leu-Gln-Ala-Arg-Leu-Asp-Arg-Leu-Leu-Arg-Arg-Leu-Gln-Leu-Leu-Met-Ser-Arg-Leu-Ala-Leu-Pro-Gln-Pro-Pro-Pro-Asn-Pro-Pro-Ala-Pro-Pro-Leu-Ala-Pro-Pro-Ser-Ser-Ala-Trp-Gly-Gly-Ile-Arg-Ala-Ala-His-Ala-Ile-Leu-Gly-Gly-Leu-His-Leu-Thr-Leu-Asp-Trp-Ala-Val-Arg-Gly-Leu-Leu-Leu-Leu-Lys-Thr-Arg-Leu

The expression plasmid was transformed into E. coli BL21.

Transformants were inoculated in LB Broth supplemented with ampicillin(50 μg/ml final concentration), and incubated at 37° C. until OD 600reached 0.5, and IPTG was added to a final concentration of 0.4 mM.Incubation was continued for 3 h, and the cells were harvested bycentrifugation. The expression of GST-IL11(LV−) was confirmed bySDS-PAGE. The bacterial pellets were resuspended in 50 mM Tris-HClbuffer, pH 8.0 and lysed by sonication. GST-IL11(LV−) was purified byGlutathione Sepharose 4 Fast Flow from the soluble fraction.

The purified fusion proteins were digested with thrombin and analyzed bySDS-PAGE. One hundred micrograms of protein samples were subjected tocleavage by 0.5 units thrombin in 25 mM Tris-HCl buffer containing 150mM NaCl, pH 8, at 20° C. Aliquots were removed from each reaction atvarious time points (10 min, 30 min, 1 h, 2 h, and 3 h after reaction),and heat-inactivated by boiling for 5 min to stop the reaction. Thefusion protein GST-IL11(LV−) (44 kDa) gradually disappeared, and theamount of IL-11(LV−) (18 kDa) increased as reaction time passed (FIG.11).

To check where the cleavage occurred by thrombin treatment, the proteinband containing IL-11(LV−) protein was obtained, and subjected to aminoacid sequencing. First, the purified GST-IL11(LV−) protein was treatedwith thrombin for 3 h, and separated by SDS-PAGE. Then, the proteins inthe polyacrylamide gel were transferred to PVDF membrane. After the bandcontaining IL-11(LV−) protein was identified, it was sliced away fromthe PVDF membrane, and subjected to amino acid sequencing. It wasconfirmed that the IL-11(LV−) has the sequenceAla-Ser-Pro-Asp-Pro-Arg-Ala-Glu-Leu-Asp (SEQ ID NO:35) at itsN-terminus, suggesting that the Pro-Arg-Gly-Ser-Pro-Arg-Ala-Ser (SEQ IDNO:7) sequence is enough for efficient thrombin cleavage.

Example 6 MBP-IL11

The MBP-IL11 expression plasmid, pMAL-c2-IL11, was constructed byinserting a cDNA of human IL-11 into pMAL-c2 (FIG. 12). A cDNA encodinghuman Th-11 was obtained from pGEX4T-IL11(A) by PCR. The oligonucleotidesequences for PCR were as follows:

(SEQ ID NO: 36) 5′: GAA TTC CCT CGA GGT TCA CCT CGA GCT TCC EcoRI (SEQID NO: 37) 3′: GTC GAC TCA CAG CCG AGT CTT CAG CAG SalI

The 5′ primer was designed to contain the coding sequence forPro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:7). The EcoRI/SalI fragmentcontaining IL-11 sequence was cloned into the EcoRI/SalI site of thepMAL-c2 vector, producing pMAL-c2-IL11. Therefore, pMAL-c2-IL11 has thesame thrombin cleavage site as pGEX-IL11(LV−) between the maltosebinding domain and human IL-11 sequences.

The amino acid sequence of IL-11 downstream of the maltose bindingdomain including the thrombin cleavage site is given below.

(SEQ ID NO: 34)Pro-Arg-Gly-Ser-Pro-Arg↓-Ala-Ser-Pro-Asp-Pro-Arg-Ala-Glu-Leu-Asp-Ser-Thr-Val-Leu-Leu-Thr-Arg-Ser-Leu-Leu-Ala-Asp-Thr-Arg-Gln-Leu-Ala-Ala-Gln-Leu-Arg-Asp-Lys-Phe-Pro-Ala-Asp-Gly-Asp-His-Asn-Leu-Asp-Ser-Leu-Pro-Thr-Leu-Ala-Met-Ser-Ala-Gly-Ala-Leu-Gly-Ala-Leu-Gln-Leu-Pro-Gly-Val-Leu-Thr-Arg-Leu-Arg-Ala-Asp-Leu-Leu-Ser-Tyr-Leu-Arg-His-Val-Gln-Trp-Leu-Arg-Arg-Ala-Gly-Gly-Ser-Ser-Leu-Lys-Thr-Leu-Glu-Pro-Glu-Leu-Gly-Thr-Leu-Gln-Ala-Arg-Leu-Asp-Arg-Leu-Leu-Arg-Arg-Leu-Gln-Leu-Leu-Met-Ser-Arg-Leu-Ala-Leu-Pro-Gln-Pro-Pro-Pro-Asn-Pro-Pro-Ala-Pro-Pro-Leu-Ala-Pro-Pro-Ser-Ser-Ala-Try-Gly-Gly-Ile-Arg-Ala-Ala-His-Ala-Ile-Leu-Gly-Gly-Leu-His-Leu-Thr-Leu-Asp-Trp-Ala-Val-Arg-Gly-Leu-Leu-Leu-Leu-Lys-Thr-Arg-Leu

The expression plasmid was transformed into E. coli BL21. Transformantswere inoculated in LB Broth supplemented with ampicillin (50 μg/ml finalconcentration), and incubated at 37° C. until OD 600 reached 0.5, andIPTG was added to a final concentration of 0.4 mM. Incubation wascontinued for 3 h, and the cells were harvested by centrifugation. Theexpression of MBP-IL11 was confirmed by SDS-PAGE. The bacterial pelletswere resuspended in 20 mM Tris-HCl buffer, pH 7.4 containing 200 mMNaCl, 1 mM EDTA, and 1 mM sodium azide and lysed by sonication. Thesupernatant containing target protein was applied to an amylose (NewEngland Biolabs) affinity column pre-equilibrated with 20 mM Tris-HClbuffer, pH 7.4 containing 200 mM NaCl, and 1 mM EDTA. Following severalwashes, the bound fusion protein was eluted with 20 mM Tris-HCl buffer,pH 7.4 containing 200 mM NaCl, 1 mM EDTA, and 10 mM maltose.

The purified fusion proteins were digested with thrombin and analyzed bySDS-PAGE. One hundred micrograms of protein samples were subjected tocleavage by 0.5 units thrombin in 20 mM Tris-HCl buffer, pH 7.4containing 200 mM NaCl, 1 mM EDTA, and 10 mM maltose, at 20° C. Aliquotswere removed from each reaction at various time points (10 min, 30 min,1 h, 2 h, and 3 h after reaction), and heat-inactivated by boiling for 5min to stop the reaction. The fusion protein MBP-IL11 (60 kDa) graduallydisappeared, and the amount of IL11 (18 kDa) increased as reaction timepassed (FIG. 13).

To check where the cleavage occurred by thrombin treatment, the proteinband containing IL-11 protein was obtained, and subjected to amino acidsequencing. First, the purified MBP-IL11 protein was treated withthrombin for 3 h, and separated by SDS-PAGE. Then, the proteins in thepolyacrylamide gel were transferred to PVDF membrane. After the bandcontaining IL-11 protein was identified, it was sliced away from thePVDF membrane, and subjected to amino acid sequencing. It was confirmedthat the IL-11 has the sequence Ala-Ser-Pro-Asp-Pro-Arg-Ala-Glu-Leu-Asp(SEQ ID NO:38) at its N-terminus, suggesting that thePro-Arg-Gly-Ser-Pro-Arg-Ala-Ser (SEQ ID NO:7) sequence is enough forefficient thrombin cleavage.

Example 7 HIS-IL11

The His-IL11 expression plasmid, pET19b-IL11, was constructed byinserting a cDNA of human IL-11 into pET-19b (FIG. 14). A cDNA encodinghuman IL-11 was obtained from pGEX4T-IL11(A) by PCR. The oligonucleotidesequences for PCR were as follows:

(SEQ ID NO: 39) 5′: CAT ATG CCT CGA GGT TCA CCT CGA GCT TCC NdeI (SEQ IDNO: 40) 3′: GGA TCC TCA CAG CCG AGT CTT CAG CAG BamHI

The 5′ primer was designed to contain the coding sequence forPro-Arg-Gly-Ser-Pro-Arg↓Ala-Ser (SEQ ID NO:7). The NdeI/BamHI fragmentcontaining IL-11 sequence was cloned into the NdeI/BamHI site of thepET19b vector, generating pET19b-IL11. Therefore, pET19b-IL11 has thesame thrombin cleavage site as pGEX-IL11(LV−) between the His tag andhuman IL-11 sequences.

The amino acid sequence of IL-11 downstream of the His tag including thethrombin cleavage site is given below.

(SEQ ID NO: 34)Pro-Arg-Gly-Ser-Pro-Arg↓-Ala-Ser-Pro-Asp-Pro-Arg-Ala-Glu-Leu-Asp-Ser-Thr-Val-Leu-Leu-Thr-Arg-Ser-Leu-Leu-Ala-Asp-Thr-Arg-Gln-Leu-Ala-Ala-Gln-Leu-Arg-Asp-Lys-Phe-Pro-Ala-Asp-Gly-Asp-His-Asn-Leu-Asp-Ser-Leu-Pro-Thr-Leu-Ala-Met-Ser-Ala-Gly-Ala-Leu-Gly-Ala-Leu-Gln-Leu-Pro-Gly-Val-Leu-Thr-Arg-Leu-Arg-Ala-Asp-Leu-Leu-Ser-Tyr-Leu-Arg-His-Val-Gln-Trp-Leu-Arg-Arg-Ala-Gly-Gly-Ser-Ser-Leu-Lys-Thr-Leu-Glu-Pro-Glu-Leu-Gly-Thr-Leu-Gln-Ala-Arg-Leu-Asp-Arg-Leu-Leu-Arg-Arg-Leu-Gln-Leu-Leu-Met-Ser-Arg-Leu-Ala-Leu-Pro-Gln-Pro-Pro-Pro-Asn-Pro-Pro-Ala-Pro-Pro-Leu-Ala-Pro-Pro-Ser-Ser-Ala-Try-Gly-Gly-Ile-Arg-Ala-Ala-His-Ala-Ile-Leu-Gly-Gly-Leu-His-Leu-Thr-Leu-Asp-Trp-Ala-Val-Arg-Gly-Leu-Leu-Leu-Leu-Lys-Thr-Arg-Leu

The expression plasmid was transformed into E. coli BL21(DE3).Transformants were inoculated in LB Broth supplemented with ampicillin(50 μg/ml final concentration), and incubated at 37° C. until OD 600reached 0.5, and IPTG was added to a final concentration of 1 mM.Incubation was continued for 3 h, and the cells were harvested bycentrifugation. The expression of His-IL11 was confirmed by SDS-PAGE.The bacterial pellets were resuspended in 20 mM sodium phosphate buffer,pH 7.4 containing 0.5 M NaCl and lysed by sonication. The supernatantcontaining target protein was applied to a Ni-Sepharose High Performance(Amersham Biosciences) affinity column pre-equilibrated with 20 mMsodium phosphate buffer, pH 7.4 containing 0.5 M NaCl and 20 mMimidazole. Following several washes, the bound fusion protein was elutedwith 20 mM sodium phosphate buffer, pH 7.4 containing 0.5 M NaCl and 500mM imidazole.

The purified fusion proteins were digested with thrombin and analyzed bySDS-PAGE. One hundred micrograms of protein samples were subjected tocleavage by 0.5 units thrombin in 20 mM sodium phosphate buffer, pH 7.4containing 0.5 M NaCl and 500 mM imidazole, at 20° C. Aliquots wereremoved from each reaction at various time points (10 min, 30 min, 1 h,2 h, and 3 h after reaction), and heat-inactivated by boiling for 5 minto stop the reaction. The fusion protein His-IL11 (22 kDa) graduallydisappeared, and the amount of IL11 (18 kDa) increased as reaction timepassed (FIG. 15).

To check where the cleavage occurred by thrombin treatment, the proteinband containing IL-11 protein was obtained, and subjected to amino acidsequencing. First, the purified His-IL11 protein was treated withthrombin for 3 h, and separated by SDS-PAGE. Then, the proteins in thepolyacrylamide gel were transferred to PVDF membrane. After the bandcontaining IL-11 protein was identified, it was sliced away from thePVDF membrane, and subjected to amino acid sequencing. It was confirmedthat the IL-11 has the sequence Ala-Ser-Pro-Asp-Pro-Arg-Ala-Glu-Leu-Asp(SEQ ID NO:41) at its N-terminus, suggesting that thePro-Arg-Gly-Ser-Pro-Arg-Ala-Ser (SEQ ID NO:7) sequence is enough forefficient thrombin cleavage.

Having now fully described the invention, it will be understood by thoseof ordinary skill in the art that the same can be performed within awide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

1-17. (canceled)
 18. A method of separating the protein of interest fromthe chimeric protein of claim 35 comprising contacting the chimericprotein with a sufficient amount of thrombin such that cleavage of thepeptide linker occurs.
 19. The method of claim 18, wherein said chimericprotein is cleaved between X3 and X4 of the peptide linker.
 20. Apolynucleotide encoding the chimeric protein of claim
 35. 21-25.(canceled)
 26. A vector comprising the polynucleotide of claim
 20. 27. Ahost cell comprising the vector of claim
 26. 28. A method of producing achimeric protein, comprising: a) preparing a vector comprising apolynucleotide encoding a chimeric protein of claim 35; b) deliveringsaid vector into a host cell; c) culturing said host cell underconditions in which the chimeric protein is expressed; and d) separatingsaid chimeric protein.
 29. A method of producing a protein of interest,comprising: a) preparing a vector comprising a polynucleotide encoding achimeric protein of claim 35; b) delivering said vector into a hostcell; c) culturing said host cell under conditions in which the chimericprotein is expressed; d) separating said chimeric protein; e) contactingsaid chimeric protein with thrombin to cleave said chimeric protein; andd) separating said protein of interest.
 30. The method of claim 29,wherein said separating of said chimeric protein is performed with anaffinity column.
 31. The method of claim 30, wherein said contacting ofsaid chimeric protein with thrombin is performed while said chimericprotein is bound on said affinity column.
 32. The method of claim 30,wherein said separating of said chimeric protein further comprisessubjecting said protein of interest to cation exchange chromatography.33. The method of claim 30, wherein said separating of said chimericprotein further comprises subjecting said protein of interest to anionexchange chromatography.
 34. A kit comprising the vector of claim 26.35. A chimeric protein comprising a protein of interest, a fusionpartner, and a peptide linker interposed there between, wherein saidprotein of interest is linked to the N-terminus of said peptide linkerand said fusion partner is linked to the C-terminus of said peptidelinker or said protein of interest is linked to the C-terminus of saidpeptide linker and said fusion partner is linked to the N-terminus ofsaid peptide linker, wherein said peptide linker consists of thesequence: X1-X2-Ser-Pro-X3-X4-X5 wherein, X1 is two to ten amino acidresidues that are the same or different from each other; X2 is glycine;X3 is arginine or lysine; X4 is alanine or glycine; and X5 is anon-acidic amino acid, wherein (a) said fusion partner is an affinitypeptide; (b) said protein of interest is selected from the groupconsisting of human interleukin (IL)-11, thymosin β4, thymosin α1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, IL-15, IL-18,Protease-activated receptor 1 (PAR1), PAR3, PAR4, RANTES, stromalcell-derived factor-1α, monocyte chemotactic protein, stem cell factor,FLT-3L, parathyroid hormone, thrombopoietin, epidermal growth factor,basic fibroblast growth factor, insulin-like growth factor,granulocyte-macrophage colony stimulating factor, granulocyte colonystimulating factor, macrophage colony stimulating factor,platelet-derived growth factor, transforming growth factor (TGF)-β1,tumor necrosis factor (TNF)-α, interferon (IFN)-α, IFN-γ, hepatocytegrowth factor, vascular endothelial growth factor and immunoglobulinheavy chain, wherein said chimeric protein is non-naturally occurring;or (c) said fusion partner is an affinity peptide and said protein ofinterest is selected from the group consisting of human interleukin(IL)-11, thymosin β4, thymosin α1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-13, IL-15, IL-18, Protease-activated receptor 1 (PAR1),PAR3, PAR4, RANTES, stromal cell-derived factor-1α, monocyte chemotacticprotein, stem cell factor, FLT-3L, parathyroid hormone, thrombopoietin,epidermal growth factor, basic fibroblast growth factor, insulin-likegrowth factor, granulocyte-macrophage colony stimulating factor,granulocyte colony stimulating factor, macrophage colony stimulatingfactor, platelet-derived growth factor, transforming growth factor(TGF)-β1, tumor necrosis factor (TNF)-α, interferon (IFN)-α, IFN-β,IFN-γ, hepatocyte growth factor, vascular endothelial growth factor andimmunoglobulin heavy chain.
 36. The chimeric protein of claim 35,wherein X1 is four to ten amino acid residues.
 37. The chimeric proteinof claim 36, wherein X1 comprises proline and arginine.
 38. The chimericprotein of claim 35, wherein X5 is selected from the group consisting ofserine, alanine, asparagine, valine, leucine, isoleucine, lysine,phenylalanine, tyrosine, and tryptophan.
 39. (canceled)
 40. The chimericprotein of claim 35, wherein said affinity peptide is selected from thegroup consisting of glutathione-S-transferase (GST), maltose bindingprotein (MBP), hexahistidine, T7 peptide, ubiquitin, Flag peptide, c-mycpeptide, polyarginine, polycysteine, polyphenylalanine, BTag, galactosebinding domain, cellulose binding domain (CBD), thioredoxin,staphylococcal protein A, streptococcal protein G, calmodulin,beta-galactosidase, chloramphenicol acetyltransferase, S-peptide,streptavidin, His-tag, and Strep-tag. 41-42. (canceled)
 43. The chimericprotein of claim 35, wherein said protein of interest is selected fromthe group consisting of human IL11, thymosin β4, IL-6 and PAR4.
 44. Thechimeric protein of claim 35, wherein said protein of interest is linkedto the N-terminus of said peptide linker and said fusion partner islinked to the C-terminus of said peptide linker.
 45. The chimericprotein of claim 35, wherein said protein of interest is linked to theC-terminus of said peptide linker and said fusion partner is linked tothe N-terminus of said peptide linker. 46-60. (canceled)